Our principal objective is an immunochemical approach to unresolved problems of receptor structure, localization and function that is now possible with our variety of cross-reacting and species-specific estrophilin antibodies. Such studies include purification of estrophilin by immunoadsorption on immobilized monoclonal antibody, development of simple radioimmunoassay procedures for measuring receptors in normal and neoplastic tissues, and precise intracellular localization of estrophilin by techniques of immunohistology combined with electron microscopy. Since detection of receptor by antibody does not depend on its binding to steroid, we shall study intracellular receptor distribution before, as well as after, its perturbation by exposure to estrogen, search for a possible non-binding receptor precursor (proestrophilin), and determine the fate of translocated receptor protein in the nucleus after it has parted company with the hormone. In collaboration with other investigators who possess the appropriate expertise we shall: (1) study the interaction of the different antibodies with mero receptor and other proteolytic fragments to obtain clues to the molecular location of conserved as compared to species-specific antigenic determinants; (2) investigate the immunochemical similarity of type I and type II binding components of target tissues; and (3) use the antibody to identify estrophilin-producing clones of recombinant bacterial plasmids after insertion of cDNA, prepared from poly A-containing mRNAs from receptor-rich tissues, in an attempt to develop a plasmid system that synthesizes receptor in large quantity and that could furnish appropriate DNA fragments from whose nucleotide pattern the amino acid sequence of the receptor can be deduced. In addition to immunochemical studies, we will continue our investigations of the use of CPG-beads in a simple method for measuring estrogen and progesterone receptors in tissue and tumor cytosols and in estimating occupied nuclear receptors by Ag(I)-mediated exchange at low temperature. A promising, novel affinity chromatography system for estrophilin purification will be further explored. Once a dependable procedure for removing the steroid from hormone-activated estrophilin is developed, the uncomplexed receptor protein will be compared with its estradiol complex in its ability to stimulate RNA polymerase activity in isolated uterine nuclei.